Bottom Line:
As a result, only the latter underwent a rapid and profound acidification.The paucity of V-ATPases in M1 phagosomes was associated with, and likely caused by, delayed fusion with late endosomes and lysosomes.By contrast, M2 phagosomes proceed to acidify immediately in order to clear apoptotic bodies rapidly and effectively.

Figure 6: Phagosomal pH oscillations in M1 macrophages. (A) M1 macrophages were challenged with SNARF1-SOZ, and, upon particle uptake, pH measurements were acquired every minute for 30 min. The leftmost trace represents the mean ± SEM of 16 determinations. Three individual experiments are shown to the right of the averaged trace, to highlight the characteristic pH oscillations. (B) M1 macrophages were challenged with SNARF1-SOZ in the presence of 100 μM Zn2+. The leftmost trace represents the mean ± SEM of eight determinations. Three individual experiments are shown to the right of the averaged trace.

Mentions:
Whereas the phagosomal pH in M1 macrophages was routinely more alkaline than that of M2 macrophages, it was not constant over time. The variability, which is discernible in the averaged results illustrated in Figure 1A (replicated for reference in Supplemental Figure S4A), becomes more manifest when individual experiments are displayed (Supplemental Figure S4A). Such pH oscillations were unique to M1 phagosomes, as they were never observed in M2 phagosomes (Supplemental Figure S5A). Because fluorescein is best suited for pH measurements near its pKa (6.3), we confirmed the occurrence of oscillations near and above neutral pH using SOZ covalently labeled with SNARF1 (pKa = 7.5), a fluorophore better suited for pH determinations in the alkaline range (Figure 6A).

Figure 6: Phagosomal pH oscillations in M1 macrophages. (A) M1 macrophages were challenged with SNARF1-SOZ, and, upon particle uptake, pH measurements were acquired every minute for 30 min. The leftmost trace represents the mean ± SEM of 16 determinations. Three individual experiments are shown to the right of the averaged trace, to highlight the characteristic pH oscillations. (B) M1 macrophages were challenged with SNARF1-SOZ in the presence of 100 μM Zn2+. The leftmost trace represents the mean ± SEM of eight determinations. Three individual experiments are shown to the right of the averaged trace.

Mentions:
Whereas the phagosomal pH in M1 macrophages was routinely more alkaline than that of M2 macrophages, it was not constant over time. The variability, which is discernible in the averaged results illustrated in Figure 1A (replicated for reference in Supplemental Figure S4A), becomes more manifest when individual experiments are displayed (Supplemental Figure S4A). Such pH oscillations were unique to M1 phagosomes, as they were never observed in M2 phagosomes (Supplemental Figure S5A). Because fluorescein is best suited for pH measurements near its pKa (6.3), we confirmed the occurrence of oscillations near and above neutral pH using SOZ covalently labeled with SNARF1 (pKa = 7.5), a fluorophore better suited for pH determinations in the alkaline range (Figure 6A).

Bottom Line:
As a result, only the latter underwent a rapid and profound acidification.The paucity of V-ATPases in M1 phagosomes was associated with, and likely caused by, delayed fusion with late endosomes and lysosomes.By contrast, M2 phagosomes proceed to acidify immediately in order to clear apoptotic bodies rapidly and effectively.